Bicinchoninic acid protein concentration assay kit

Total protein from liver tissue was extracted and quantified with a bicinchoninic acid protein concentration assay kit (Thermo Scientific) and then separated by 12% SDS/PAGE with a Bio-Rad electrophoresis system. Blotted membranes were incubated for 1 h with blocking solution (Tris-buffered saline/0.1% Tween 20, TBST) containing 5% skim milk (w/v) at room temperature. Subsequently, the membrane was incubated overnight at 4 °C or 1 h at room temperature with primary antibody [anti-granzyme B (catalog: A2557), anti-perforin (catalog: A0093), TRAIL (catalog: A12064), and anti-Caspase-3 (catalog: A19654) polyclonal antibody from Abclonal (Wuhan, China); and anti-Caspase-8 polyclonal antibody (catalog: 13423-1-AP) and anti-GAPDH monoclonal antibody (catalog: 60004-1-Ig) from Proteintech (Wuhan, China)]. After being washed, the membrane was incubated 1 h with 1:10,000 diluted HRP-conjugated polyclonal goat anti-rabbit IgG (H+L) or HRP-conjugated polyclonal goat anti-mouse IgG (H+L) antibodies (Proteintech, China). The membranes were washed, and the immunoblot was then developed with an ECL chemiluminescence detection kit (Thermo Scientific) according to the manufacturer’s instructions. To normalize for protein loading, GAPDH was used, and the protein expression levels were calculated relative to the value of GAPDH.

Cell pellets from metabolite extraction were resuspended in 5% SDS (w/v) and 0.1 M NaOH, and protein concentration was estimated using the bicinchoninic acid protein concentration assay kit (Thermo Fisher Scientific) following the manufacturer’s instructions. External protein standards were analyzed alongside the samples to estimate protein amounts.